Enzyme-linked immunosorbent assay with antigen-coated plates

Even more popular than dot blots are microtiter plate assays, so-called ELlSAs (enzyme-linked immunosorbent assay), in which antibody or antigen is loaded into the depression of polyvinyl chloride or polystyrene plates (Kemeny 1994). The depressions are then further coated with antibody, antigen, and enzyme-conjugated antibody in a defined sequence. The antigen is detected via an enzymatic color reaction (Table 6.2). Many companies (Nunc, Flow, Costar, Falcon) offer a palette of products such as 8- or 12-channel pipettes, automatic washing devices, ELISA readers, and so on that make life easier for the friends of ELISA. 

Competition Enzvme-Linked Immunosorbent Assay. A competition enzyme-linked immunosorbent assay (cELISA) was developed to quantify the amount of heptachlor in solution and to evaluate the ability of the antibodies to distinguish among various cyclodiene insecticides and related chemicals. Microtiter plates were coated with 0.25 ng/well hept-BSA (100 p.l/well of a 2.5 ng/mL solution of hept-BSA in distilled water was allowed to evaporate onto the bottoms of the wells at 37° C). The plates were then blocked with DB as described above. Competitors (analytical standards dissolved in methanol) were added to the DB such that the resulting solution was 50% methanol. An aliquot (200 pL) of this competitor-dilution buffer solution was added to an antigen-coated well. Then, the amount of competitor was serially diluted down the microtiter plate, so each well contained 100 iL of competitor in a 50% methanol-dilution buffer solution. Next an equal volume of dilution DB 100 ng of anti-heptachlor monoclonal antibody was added to each well. Thus, each well contained 200 )xl of a 25% methanol solution in DB, antibody and competitor. Plates were incubated for 1 h at 37° C and then processed as described above. 

Bioassay-based or enzyme-linked immunosorbent assay (ELISA) methods have been applied in epitope mapping [18, 19]. In this approach, different antigens are coated separately on the surface in different weUs of the analytical plate followed by incubation with a specific antibody that is linked to an enzyme. When the enzyme s substrate is added to the solution, a subsequent reaction can produce a detectable signal. This approach, however, is mostly limited to the characterization of linear epitopes. ELISA can also be applied to multisite binding analysis, where one antibody is attached to the surface, the antigen is bound, and the ability of the second antibody to bind to the attached antibody-antigen... 

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